Abstract Bone fractures, due to aging and disease, contribute significantly to healthcare cost and affect the quality of life with aging and disease. Non-enzymatic glycation (NEG) is a post-translational modification process by sugars on long-lived proteins. In bone, NEG's effects on bone matrix proteins are associated with type 2 diabetes mellitus (T2D) and linked to bone fragility fractures. NEG results in the accumulation of fluorescent and non-fluorescent advanced glycation end- products (AGEs). Currently, pentosidine (PEN) is the only established measure of NEG in bone. However, it represents only a small fraction of fluorescent AGEs (fAGEs). Thus, the extent of NEG in bone and how it influences bone fragility remains unknown. Here we demonstrate, for the first time, the formation of carboxy-methyl lysine (CML) in bone and modification of the collagen-hydroxyapatite (HA) distance by AGEs. CML is a non- fluorescent AGE that has a short metabolic pathway and can form in more significant amounts than other AGEs, influencing bone fragility. In the previous grant cycle, we showed that cancellous bone accumulates more AGEs than cortical bone. Here we show that the amount of AGEs in cancellous bone and cortical shell associate differently with pre- and post- yield mechanical properties of whole vertebra. Accumulation of AGEs in cortical and cancellous bone may therefore better explain whole bone fractures. We also show increased accumulation of AGEs and loss of fracture toughness using a high fat diet rat model which simulates T2D in humans without the confound of genetic mutations. Furthermore, AGEs in bone can be cleaved by phenacylthiazolium-based compounds to rescue bone fragility. Thus our overall goal is to determine the effects of AGEs and their removal on diabetic and fragility fractures. Using a combination of in vitro, ex vivo and in vivo models we will pursue the following aims: Aim 1: To establish the proportion of CML, PEN and fAGEs in cortical and cancellous bone and determine whether accumulation of AGEs causes the modification of the collagen-HA interface; Aim 2: To determine the contribution of cortical and cancellous bone AGEs on whole bone fragility and evaluate whether their removal rescues bone fragility in an ex vivo human model; Aim 3: To establish the role of AGEs in causing increased bone fragility in T2D, using a validated diet-induced rat model, and to test whether removal of AGEs and rescue T2D bone fragility. Our findings will lead to the development of novel modalities for determining fracture risk, new understanding of T2D and fragility fractures, and new approaches to control skeletal fragility by managing AGEs.